We have developed a new approach to facilitate identification of natural products with antibacterial activities that rapidly discriminates between different mechanisms of action (MOA). This approach, bacterial cytological profiling (BCP), uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely MOA of new compounds within a few hours. The approach can detect nuisance compounds and multiple activities in partially purified or crude natural product extracts and it can detect active molecules at sub-inhibitory concentrations. Linnaeus Bioscience Inc. is a start up company founded to commercialize this technology and make it accessible on a fee for service basis to the pharmaceutical industry and the scientific community. The goal of this project is to make critical improvements to BCP technology that will allow it to realize its potential in the drug discovery pipeline to more rapidly characterize, prioritize and identify lead molecules. Specifically, we propose the following research aims: (1) Improve the throughput of BCP by ten fold so that we can process more than 200 samples per day. (2) Create a comprehensive reference set of cytological profiles that will allow us to quickly and accurately pinpoint the most likely MOA of new compounds. (3) Extend BCP to multidrug resistant bacteria so that it can be more widely applied. (4) Test the utility of BCP as a primary screen for antibacterial activities by screening library of 200 previously unscreened diverse natural product extracts. (5) Test the utility of BCP in a screen for compounds that synergize with the antibiotic colistin. These studies will demonstrate that BCP is a robust whole cell and imaging based method capable of identifying molecules with antibacterial properties from natural product extracts on an industrial scale. This platform will be made widely available on a fee for service basis.